Polypeptides having antimicrobial activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having antimicrobial activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/271,045filed Nov. 11, 2005, now U.S. Pat. No. 7,388,077, which claims priorityor the benefit under 35 U.S.C. 119 of Danish application no. PA 200401744 filed Nov. 12, 2004 and U.S. provisional application No.60/629,442 filed Nov. 19, 2004, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to isolated polypeptides havingantimicrobial activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods for producing and using the polypeptides.

BACKGROUND OF THE INVENTION

It is an object of the present invention to provide polypeptides havingantimicrobial activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingantimicrobial activity selected from the group consisting of:

-   -   (a) a polypeptide having an amino acid sequence which has at        least 60% identity with amino acids 1 to 42 of SEQ ID NO:2;    -   (b) a polypeptide which is encoded by a nucleotide sequence        which hybridizes under at least medium stringency conditions        with (i) nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNA        sequence contained in nucleotides 1 to 270 of SEQ ID NO:1,        or (iii) a complementary strand of (i) or (ii); and    -   (c) a variant comprising a conservative substitution, deletion,        and/or insertion of one or more amino acids of amino acids 1 to        42 of SEQ ID NO:2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having antimicrobial activity, selected from the groupconsisting of:

-   -   (a) a polynucleotide encoding a polypeptide having an amino acid        sequence which has at least 60% identity with amino acids 1 to        42 of SEQ ID NO:2;    -   (b) a polynucleotide having at least 60% identity with        nucleotides 145 to 270 of SEQ ID NO:1, and    -   (c) a polynucleotide which hybridizes under at least medium        stringency conditions with (i) nucleotides 145 to 270 of SEQ ID        NO:1, (ii) the cDNA sequence contained in nucleotides 1 to 270        of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii).

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides.

The present invention also relates to methods for producing suchpolypeptides having antimicrobial activity comprising (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of using the polypeptidesand polynucleotides of the invention.

DEFINITIONS

Antimicrobial activity: The term “antimicrobial activity” is definedherein as an activity which is capable of killing or inhibiting growthof microbial cells. In the context of the present invention the term“antimicrobial” is intended to mean that there is a bactericidal and/ora bacteriostatic and/or fungicidal and/or fungistatic effect and/or avirucidal effect, wherein the term “bactericidal” is to be understood ascapable of killing bacterial cells. The term “bacteriostatic” is to beunderstood as capable of inhibiting bacterial growth, i.e. inhibitinggrowing bacterial cells. The term “fungicidal” is to be understood ascapable of killing fungal cells. The term “fungistatic” is to beunderstood as capable of inhibiting fungal growth, i.e. inhibitinggrowing fungal cells. The term “virucidal” is to be understood ascapable of inactivating virus. The term “microbial cells” denotesbacterial or fungal cells (including yeasts).

In the context of the present invention the term “inhibiting growth ofmicrobial cells” is intended to mean that the cells are in thenon-growing state, i.e., that they are not able to propagate.

For purposes of the present invention, antimicrobial activity may bedetermined according to the procedure described by Lehrer et al.,Journal of Immunological Methods, Vol. 137 (2) pp. 167-174 (1991).Alternatively, antimicrobial activity may be determined according to theNCCLS guidelines from CLSI (Clinical and Laboratory Standards Institute;formerly known as National Committee for Clinical and LaboratoryStandards).

Polypeptides having antimicrobial activity may be capable of reducingthe number of living cells of Escherichia coli (DSM 1576) to 1/100 after8 hours (preferably after 4 hours, more preferably after 2 hours, mostpreferably after 1 hour, and in particular after 30 minutes) incubationat 20° C. in an aqueous solution of 25% (w/w); preferably in an aqueoussolution of 10% (w/w); more preferably in an aqueous solution of 5%(w/w); even more preferably in an aqueous solution of 1% (w/w); mostpreferably in an aqueous solution of 0.5% (w/w); and in particular in anaqueous solution of 0.1% (w/w) of the polypeptides having antimicrobialactivity.

Polypeptides having antimicrobial activity may also be capable ofinhibiting the outgrowth of Escherichia coli (DSM 1576) for 24 hours at25° C. in a microbial growth substrate, when added in a concentration of1000 ppm; preferably when added in a concentration of 500 ppm; morepreferably when added in a concentration of 250 ppm; even morepreferably when added in a concentration of 100 ppm; most preferablywhen added in a concentration of 50 ppm; and in particular when added ina concentration of 25 ppm.

Polypeptides having antimicrobial activity may be capable of reducingthe number of living cells of Bacillus subtilis (ATCC 6633) to 1/100after 8 hours (preferably after 4 hours, more preferably after 2 hours,most preferably after 1 hour, and in particular after 30 minutes)incubation at 20° C. in an aqueous solution of 25% (w/w); preferably inan aqueous solution of 10% (w/w); more preferably in an aqueous solutionof 5% (w/w); even more preferably in an aqueous solution of 1% (w/w);most preferably in an aqueous solution of 0.5% (w/w); and in particularin an aqueous solution of 0.1% (w/w) of the polypeptides havingantimicrobial activity.

Polypeptides having antimicrobial activity may also be capable ofinhibiting the outgrowth of Bacillus subtilis (ATCC 6633) for 24 hoursat 25° C. in a microbial growth substrate, when added in a concentrationof 1000 ppm; preferably when added in a concentration of 500 ppm; morepreferably when added in a concentration of 250 ppm; even morepreferably when added in a concentration of 100 ppm; most preferablywhen added in a concentration of 50 ppm; and in particular when added ina concentration of 25 ppm.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the antimicrobial activity of thepolypeptide consisting of the amino acid sequence shown as amino acids 1to 42 of SEQ ID NO:2.

Defensin: The term “defensin” as used herein refers to polypeptidesrecognized by a person skilled in the art as belonging to the defensinclass of antimicrobial peptides. To determine if a polypeptide is adefensin according to the invention, the amino acid sequence ispreferably compared with the hidden markov model profiles (HMM profiles)of the PFAM database by using the freely available HMMER softwarepackage (see Example 6).

The PFAM defensin families include Defensin_(—)1 or “Mammalian defensin”(accession no. PF00323), Defensin_(—)2 or “Arthropod defensin”(accession no. PF01097), Defensin_beta or “Beta Defensin” (accession no.PF00711), Defensin_propep or “Defensin propeptide” (accession no.PF00879) and Gamma-thionin or “Gamma-thionins family” (accession no.PF00304).

The defensins may belong to the alpha-defensin class, the beta-defensinclass, the theta-defensin class, the insect (arthropod) defensin class,the plant defensin class, the mussel defensin class, or other defensinclasses wherein the amino acid sequence comprises 6 or 8 cysteines andare structurally similar to any of the before-mentioned defensinclasses. The defensins may also be synthetic defensins sharing thecharacteristic features of any of the defensin classes.

Examples of such defensins include, but are not limited to, α-DefensinHNP-1 (human neutrophil peptide) HNP-2 and HNP-3; β-Defensin-12,Drosomycin, Heliomicin, γ1-purothionin, Insect defensin A, and thedefensins disclosed in PCT applications WO 99/53053, WO 02/085934 and WO03/044049.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, at most 3%, even morepreferably at most 2%, most preferably at most 1%, and even mostpreferably at most 0.5% by weight of other polypeptide material withwhich it is natively associated. It is, therefore, preferred that thesubstantially pure polypeptide is at least 92% pure, preferably at least94% pure, more preferably at least 95% pure, more preferably at least96% pure, more preferably at least 96% pure, more preferably at least97% pure, more preferably at least 98% pure, even more preferably atleast 99%, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences may be determined by using the program FASTAincluded in version 2.0× of the FASTA program package (see W. R. Pearsonand D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448; and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA”, Methods inEnzymology 183:63-98). The scoring matrix used was BLOSUM50, gap penaltywas −12, and gap extension penalty was −2.

The degree of identity between two nucleotide sequences is determinedusing the same algorithm and software package as described above. Thescoring matrix used was the identity matrix, gap penalty was −16, andgap extension penalty was −4.

Alternatively, an alignment of two amino acid sequences is determined byusing the Needle program from the EMBOSS package (http://emboss.org)version 2.8.0. The Needle program implements the global alignmentalgorithm described in Needleman, S. B. and Wunsch, C. D. (1970) J. Mol.Biol. 48, 443-453. The substitution matrix used is BLOSUM62, gap openingpenalty is 10, and gap extension penalty is 0.5.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”; e.g. amino acids 1 to 42 of SEQ IDNO:2) and a different amino acid sequence (“foreign sequence”) iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence” or thelength of the “foreign sequence”, whichever is the shortest. The resultis expressed in percent identity.

An exact match occurs when the “invention sequence” and the “foreignsequence” have identical amino acid residues in the same positions ofthe overlap (in the alignment example below this is represented by “|”).The length of a sequence is the number of amino acid residues in thesequence (e.g. the length of amino acids 1 to 42 of SEQ ID NO:2 is 42).

In the alignment example below, the overlap is the amino acid sequence“HTWGER-NL” of Sequence 1; or the amino acid sequence “HGWGEDANL” ofSequence 2. In the example a gap is indicated by a “-”.

Alignment Example

Polypeptide Fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of SEQ ID NO:2 or a homologous sequencethereof, wherein the fragment has antimicrobial activity. Preferably apolypeptide fragment of the invention retains all cysteine residues andthe amino acid residues between the cysteine residues.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of SEQ ID NO:1 or a homologous sequence thereof, wherein thesubsequence encodes a polypeptide fragment having antimicrobialactivity.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may a DNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct comprising a polynucleotide ofthe present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the amino acids 1 to 42 ofSEQ ID NO:2 as well as genetic manipulation of the DNA encoding thatpolypeptide. The modification(s) can be substitution(s), deletion(s)and/or insertions(s) of the amino acid(s) as well as replacement(s) ofamino acid side chain(s); or use of unnatural amino acids with similarcharacteristics in the amino acid sequence. In particular themodification(s) can be amidations, such as amidation of the C-terminus.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having antimicrobial activity produced by anorganism expressing a modified nucleotide sequence of SEQ ID NO:1. Themodified nucleotide sequence is obtained through human intervention bymodification of the nucleotide sequence disclosed in SEQ ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having AntimicrobialActivity

In a first aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 1 to 42 of SEQ ID NO:2 (i.e., the maturepolypeptide) of at least 60%, preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 97%,which have antimicrobial activity (hereinafter “homologouspolypeptides”). In a preferred aspect, the homologous polypeptides havean amino acid sequence which differs by ten amino acids, preferably byfive amino acids, more preferably by four amino acids, even morepreferably by three amino acids, most preferably by two amino acids, andeven most preferably by one amino acid from amino acids 1 to 42 of SEQID NO:2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO:2 or an allelic variant thereof; or afragment thereof that has antimicrobial activity. In a preferred aspect,a polypeptide comprises the amino acid sequence of SEQ ID NO:2. Inanother preferred aspect, a polypeptide comprises amino acids 1 to 42 ofSEQ ID NO:2, or an allelic variant thereof; or a fragment thereof thathas antimicrobial activity. In another preferred aspect, a polypeptidecomprises amino acids 1 to 42 of SEQ ID NO:2. In another preferredaspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:2or an allelic variant thereof; or a fragment thereof that hasantimicrobial activity. In another preferred aspect, a polypeptideconsists of the amino acid sequence of SEQ ID NO:2. In another preferredaspect, a polypeptide consists of amino acids 1 to 42 of SEQ ID NO:2 oran allelic variant thereof; or a fragment thereof that has antimicrobialactivity. In another preferred aspect, a polypeptide consists of aminoacids 1 to 42 of SEQ ID NO:2.

In a second aspect, the present invention relates to isolatedpolypeptides having antimicrobial activity which are encoded bypolynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) nucleotides 145 to 270 of SEQ ID NO:1,(ii) the cDNA sequence contained in nucleotides 1 to 270 of SEQ ID NO:1,(iii) a subsequence of (i) or (ii), or (iv) a complementary strand of(i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.). A subsequence of SEQ ID NO:1 contains at least 100 contiguousnucleotides or preferably at least 200 continguous nucleotides.Moreover, the subsequence may encode a polypeptide fragment which hasantimicrobial activity.

The nucleotide sequence of SEQ ID NO:1 or a subsequence thereof, as wellas the amino acid sequence of SEQ ID NO:2 or a fragment thereof, may beused to design a nucleic acid probe to identify and clone DNA encodingpolypeptides having antimicrobial activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicor cDNA of the genus or species of interest, following standard Southernblotting procedures, in order to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 14, preferably at least 25, morepreferably at least 35, and most preferably at least 70 nucleotides inlength. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 270 nucleotides. BothDNA and RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having antimicrobialactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO:1 or a subsequence thereof, the carriermaterial is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO:1, itscomplementary strand, or a subsequence thereof, under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using X-ray film.

In a preferred aspect, the nucleic acid probe is a polynucleotidesequence which encodes the polypeptide of SEQ ID NO:2, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO:1. In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding region of SEQ ID NO:1.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of SEQ ID NO:2 or the mature polypeptidethereof. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,antimicrobial activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton of al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309:59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman of al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The total number of amino acid substitutions, deletions and/orinsertions of amino adds 1 to 42 of SEQ ID NO:2 is 10, preferably 9,more preferably 8, more preferably 7, more preferably at most 6, morepreferably at most 5, more preferably 4, even more preferably 3, mostpreferably 2, and even most preferably 1.

In a preferred embodiment, the polypeptides of the invention aredefensin polypeptides. In another embodiment, the polypeptides of theinvention comprise three di-cysteine bonds.

N-Terminal Extension

An N-terminal extension of the polypeptides of the invention maysuitably consist of from 1 to 50 amino acids, preferably 2-20 aminoacids, especially 3-15 amino acids. In one embodiment N-terminal peptideextension does not contain an Arg (R). In another embodiment theN-terminal extension comprises a kex2 or kex2-like cleavage site as willbe defined further below. In a preferred embodiment the N-terminalextension is a peptide, comprising at least two Glu (E) and/or Asp (D)amino acid residues, such as an N-terminal extension comprising one ofthe following sequences: EAE, EE, DE and DD.

Kex2 Sites

Kex2 sites (see, e.g., Methods in Enzymology Vol 185, ed. D. Goeddel,Academic Press Inc. (1990), San Diego, Calif., “Gene ExpressionTechnology”) and kex2-like sites are di-basic recognition sites (i.e.,cleavage sites) found between the pro-peptide encoding region and themature region of some proteins.

Insertion of a kex2 site or a kex2-like site have in certain cases beenshown to improve correct endopeptidase processing at the pro-peptidecleavage site resulting in increased protein secretion levels.

In the context of the invention insertion of a kex2 or kex2-like siteresult in the possibility to obtain cleavage at a certain position inthe N-terminal extension resulting in an antimicrobial polypeptide beingextended in comparison to the mature polypeptide shown as amino acids 1to 42 of SEQ ID NO:2.

Fused Polypeptides

The polypeptides of the present invention also include fusedpolypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide of the invention or a fragment thereof. A fused polypeptideis produced by fusing a nucleotide sequence (or a portion thereof)encoding another polypeptide to a nucleotide sequence (or a portionthereof) of the present invention. Techniques for producing fusionpolypeptides are known in the art, and include ligating the codingsequences encoding the polypeptides so that they are in frame and thatexpression of the fused polypeptide is under control of the samepromoter(s) and terminator.

Sources of Polypeptides Having Antimicrobial Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

A polypeptide of the present invention may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having antimicrobialactivity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride polypeptide.

In another preferred aspect, the polypeptide is a Eurotium amstelodami,Aspergillus amstelodami, Aspergillus montevidensis or Aspergillus vitispolypeptide.

In a more preferred aspect, the polypeptide is a Eurotium amstelodamipolypeptide, e.g., the polypeptide of SEQ ID NO:2.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of another microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encode a polypeptide of the present invention.In a preferred aspect, the nucleotide sequence is set forth in SEQ IDNO:1. In another preferred aspect, the nucleotide sequence is the maturepolypeptide coding region of SEQ ID NO:1. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof,which differ from SEQ ID NO:1 by virtue of the degeneracy of the geneticcode. The present invention also relates to subsequences of SEQ ID NO:1which encode fragments of SEQ ID NO:2 that have antimicrobial activity.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO:1, in which the mutant nucleotide sequence encodes a polypeptidewhich consists of amino acids 1 to 42 of SEQ ID NO:2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Eurotium, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO:1 (i.e., nucleotides 145 to 270) of atleast 60%, preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, even more preferably atleast 95%, and most preferably at least 97% identity, which encode anactive polypeptide.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO:1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forantimicrobial activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-enzyme interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under lowstringency conditions, preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNAsequence contained in nucleotides 1 to 270 of SEQ ID NO:1, or (iii) acomplementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under low, medium, medium-high,high, or very high stringency conditions with (i) nucleotides 145 to 270of SEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides 1 to 270of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii); and (b)isolating the hybridizing polynucleotide, which encodes a polypeptidehaving antimicrobial activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaethose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichodermareesei endoglucanase III, Trichoderma reesei endoglucanase IV,Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase); and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 60 of SEQ ID NO:1 which encode amino acids −48 to −29 of SEQ ID NO:2.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (npr7), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

In a preferred aspect, the propeptide coding region is nucleotides 61 to144 of SEQ ID NO:1 which encode amino acids −28 to −1 of SEQ ID NO:2.

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of bacterial selectable markers are the daI genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacterial, Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred aspect, theyeast host cell is a Kluyveromyces lactis cell. In another mostpreferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulaturn, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phiebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride strain cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a cell,which in its wild-type form is capable of producing the polypeptide,under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. Preferably, the cell is of the genusEurotium, and more preferably Eurotium amstelodami.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding region of SEQ IDNO:1, wherein the mutant nucleotide sequence encodes a polypeptide whichconsists of amino acids 1 to 42 of SEQ ID NO:2, and (b) recovering thepolypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies. For example, an antimicrobial activity assay maybe used to determine the activity of the polypeptide as describedherein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having antimicrobial activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the ³⁵S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen at al., 1992, Plant Mo. Biol. 18: 675-689; Zhang atal., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mot Biol. 24: 863-878), a seed specific promoter such as theglutelin, prolamin, globulin, or albumin promoter from rice (Wu et al.,1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, Journal of Plant Physiology 152: 708-711), a promoter froma seed oil body protein (Chen et al., 1998, Plant and Cell Physiology39: 935-941), the storage protein napA promoter from Brassica napus, orany other seed specific promoter known in the art, e.g., as described inWO 91/14772. Furthermore, the promoter may be a leaf specific promotersuch as the rbcs promoter from rice or tomato (Kyozuka et al., 1993,Plant Physiology 102: 991-1000, the chlorella virus adeninemethyltransferase gene promoter (Mita and Higgins, 1994, Plant MolecularBiology 26: 85-93), or the aldP gene promoter from rice (Kagaya at al.,1995, Molecular and General Genetics 248: 668-674), or a wound induciblepromoter such as the potato pint promoter (Xu at, 1993, Plant MolecularBiology 22: 573-588). Likewise, the promoter may inducible by abiotictreatments such as temperature, drought, or alterations in salinity orinduced by exogenously applied substances that activate the promoter,e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisicacid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having antimicrobial activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Compositions

The present invention also relates to compositions, such aspharmaceutical compositions, comprising a polypeptide of the presentinvention. Preferably, the compositions are enriched in such apolypeptide. The term “enriched” indicates that the antimicrobialactivity of the composition has been increased, e.g., with an enrichmentfactor of 1.1.

The compositions may further comprise another pharmaceutically activeagent, such as an additional biocidal agent, such as anotherantimicrobial polypeptide exhibiting antimicrobial activity as definedabove. The biocidal agent may be an antibiotic, as known in the art.Classes of antibiotics include penicillins, e.g. penicillin G,penicillin V, methicillin, oxacillin, carbenicillin, nafcillin,ampicillin, etc.; penicillins in combination with beta-lactamaseinhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime,moxalactam, etc.; carbapenems; monobactams; aminoglycosides;tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides;quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim;vancomycin; etc. The biocidal agent may also be an anti-mycotic agent,including polyenes, e.g. amphotericin B, nystatin; 5-flucosyn; andazoles, e.g. miconazol, ketoconazol, itraconazol and fluconazol.

In an embodiment the biocidal agent is a non-enzymatic chemical agent.In another embodiment the biocidal agent is a non-polypeptide chemicalagent.

The compositions may comprise a suitable carrier material. Thecompositions may also comprise a suitable delivery vehicle capable ofdelivering the antimicrobial polypeptides of the invention to thedesired locus when the compositions are used as a medicament.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Methods and Uses

The present invention is also directed to methods for using thepolypeptides having antimicrobial activity. The antimicrobialpolypeptides are typically useful at any locus subject to contaminationby bacteria, fungi, yeast or algae. Typically, loci are in aqueoussystems such as cooling water systems, laundry rinse water, oil systemssuch as cutting oils, lubricants, oil fields and the like, wheremicroorganisms need to be killed or where their growth needs to becontrolled. However, the present invention may also be used in allapplications for which known antimicrobial compositions are useful, suchas protection of wood, latex, adhesive, glue, paper, cardboard, textile,leather, plastics, caulking, and feed.

Other uses include preservation of foods, beverages, cosmetics such aslotions, creams, gels, ointments, soaps, shampoos, conditioners,antiperspirants, deodorants, mouth wash, contact lens products, or foodingredients.

Thus, the antimicrobial polypeptides of the invention may by useful as adisinfectant, e.g., in the treatment of infections in the eye or themouth, skin infections; in antiperspirants or deodorants; for cleaningand disinfection of contact lenses and teeth (oral care).

In general it is contemplated that the antimicrobial polypeptides of thepresent invention are useful for cleaning, disinfecting or inhibitingmicrobial growth on any surface. Examples of surfaces, which mayadvantageously be contacted with the antimicrobial polypeptides of theinvention are surfaces of process equipment used e.g. dairies, chemicalor pharmaceutical process plants, water sanitation systems, oilprocessing plants, paper pulp processing plants, water treatment plants,and cooling towers. The antimicrobial polypeptides of the inventionshould be used in an amount, which is effective for cleaning,disinfecting or inhibiting microbial growth on the surface in question.

The antimicrobial polypeptides of the invention may additionally be usedfor cleaning surfaces and cooking utensils in food processing plants andin any area in which food is prepared or served such as hospitals,nursing homes, and restaurants.

It may also be used as a preservation agent or a disinfection agent inwater based paints.

The invention also relates to the use of an antimicrobial polypeptide orcomposition of the invention as a medicament. Further, an antimicrobialpolypeptide or composition of the invention may also be used for themanufacture of a medicament for controlling or combating microorganisms,such as fungal organisms or bacteria, preferably gram positive bacteria.

The composition and antimicrobial polypeptide of the invention may beused as an antimicrobial veterinarian or human therapeutic orprophylactic agent. Thus, the composition and antimicrobial polypeptideof the invention may be used in the preparation of veterinarian or humantherapeutic agents or prophylactic agents for the treatment of microbialinfections, such as bacterial or fungal infections, preferably grampositive bacterial infections. In particular the microbial infectionsmay be associated with lung diseases including, but not limited to,tuberculosis, pneumonia and cystic fibrosis; and sexual transmitteddiseases including, but not limited to, gonorrhea and chlamydia.

The composition of the invention comprises an effective amount of theantimicrobial polypeptide of the invention.

The term “effective amount” when used herein is intended to mean anamount of the antimicrobial polypeptides of the invention, which issufficient to inhibit growth of the microorganisms in question.

The invention also relates to wound healing compositions or productssuch as bandages, medical devices such as, e.g., catheters and furtherto anti-dandruff hair products, such as shampoos.

Formulations of the antimicrobial polypeptides of the invention areadministered to a host suffering from or predisposed to a microbialinfection. Administration may be topical, localized or systemic,depending on the specific microorganism, preferably it will belocalized. Generally the dose of the antimicrobial polypeptides of theinvention will be sufficient to decrease the microbial population by atleast about 50%, usually by at least 1 log, and may be by 2 or more logsof killing. The compounds of the present invention are administered at adosage that reduces the microbial population while minimizing anyside-effects. It is contemplated that the composition will be obtainedand used under the guidance of a physician for in vivo use. Theantimicrobial polypeptides of the invention are particularly useful forkilling gram negative bacteria, including Pseudomonas aeruginosa, andChlamydia trachomatis; and gram-positive bacteria, includingstreptococci such as Streptococcus pneumonia, S. uberis, S.hyointestinalis, S. pyogenes or agalactiae; and staphylococci such asStaphylococcus aureus, S. epidermidis, S. simulans, S. xylosus, S.camosus.

Formulations of the antimicrobial polypeptides of the invention may beadministered to a host suffering from or predisposed to a microbial lunginfection, such as pneumonia; or to a microbial wound infection, such asa bacterial wound infection.

Formulations of the antimicrobial polypeptides of the invention may alsobe administered to a host suffering from or predisposed to a skininfection, such as acne, atopic dermatitis or seborrheic dermatitis;preferably the skin infection is a bacterial skin infection, e.g. causedby Staphylococcus epidermidis, Staphylococcus aureus, Propionibacteriumacnes, Pityrosporum ovale or Malassezia furfur.

The antimicrobial polypeptides of the invention are also useful for invitro formulations to kill microbes, particularly where one does notwish to introduce quantities of conventional antibiotics. For example,the antimicrobial polypeptides of the invention may be added to animaland/or human food preparations; or they may be included as an additivefor in vitro cultures of cells, to prevent the overgrowth of microbes intissue culture.

The susceptibility of a particular microbe to killing with theantimicrobial polypeptides of the invention may be determined by invitro testing, as detailed in the experimental section. Typically aculture of the microbe is combined with the antimicrobial polypeptide atvarying concentrations for a period of time sufficient to allow theprotein to act, usually between about one hour and one day. The viablemicrobes are then counted, and the level of killing determined.

Microbes of interest include, but are not limited to, Gram-negativebacteria, for example: Citrobacter sp.; Enterobacter sp.; Escherichiasp., e.g. E. coli; Klebsiella sp.; Morganella sp.; Proteus sp.;Providencia sp.; Salmonella sp., e.g. S. typhi, S. typhimurium; Serratiasp.; Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa; Yersinia sp.,e.g. Y. pestis, Y. pseudotuberculosis, Y. enterocolitica; Franciscellasp.; Pasturella sp.; Vibrio sp., e.g. V. cholerae, V. parahemolyticus;Campylobacter sp., e.g. C. jejuni; Haemophilus sp., e.g. H. influenzae,H. ducreyi; Bordetella sp., e.g. B. pertussis, B. bronchiseptica, B.parapertussis; Brucella sp., Neisseria sp., e.g. N. gonorrhoeae, N.meningitidis, etc. Other bacteria of interest include Legionella sp.,e.g. L. pneumophila; Listeria sp., e.g. L. monocytogenes; Mycoplasmasp., e.g. M. hominis, M. pneumoniae; Mycobacterium sp., e.g. M.tuberculosis, M. leprae; Treponema sp., e.g. T. pallidum; Borrelia sp.,e.g. B. burgdorferi; Leptospirae sp.; Rickettsia sp., e.g. R.rickettsii, R. typhi; Chlamydia sp., e.g. C. trachomatis, C. pneumoniae,C. psittaci; Helicobacter sp., e.g. H. pylori, etc.

Non-bacterial pathogens of interest include fungal and protozoanpathogens, e.g. Plasmodia sp., e.g. P. falciparum, Trypanosoma sp., e.g.T. brucei; shistosomes; Entaemoeba sp., Cryptococcus sp., Candida sp.,e.g. C. albicans; etc.

Various methods for administration may be employed. The polypeptideformulation may be given orally, or may be injected intravascularly,subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder,topically, etc. For example, methods of administration by inhalation arewell-known in the art. The dosage of the therapeutic formulation willvary widely, depending on the specific antimicrobial polypeptide to beadministered, the nature of the disease, the frequency ofadministration, the manner of administration, the clearance of the agentfrom the host, and the like. The initial dose may be larger, followed bysmaller maintenance doses. The dose may be administered as infrequentlyas weekly or biweekly, or fractionated into smaller doses andadministered once or several times daily, semi-weekly, etc. to maintainan effective dosage level. In many cases, oral administration willrequire a higher dose than if administered intravenously. The amidebonds, as well as the amino and carboxy termini, may be modified forgreater stability on oral administration. For example, the carboxyterminus may be amidated.

Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. More particularly, thecompounds of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, creams, foams, solutions,suppositories, injections, inhalants, gels, microspheres, lotions, andaerosols. As such, administration of the compounds can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. The antimicrobial polypeptides of the invention may besystemic after administration or may be localized by the use of animplant or other formulation that acts to retain the active dose at thesite of implantation.

In one embodiment, a formulation for topical use comprises a chelatingagent that decreases the effective concentration of divalent cations,particularly calcium and magnesium. For example, agents such as citrate,EGTA or EDTA may be included, where citrate is preferred. Theconcentration of citrate will usually be from about 1 to 10 mM.

The compounds of the present invention can be administered alone, incombination with each other, or they can be used in combination withother known compounds (e.g., perforin, anti-inflammatory agents,antibiotics, etc.) In pharmaceutical dosage forms, the compounds may beadministered in the form of their pharmaceutically acceptable salts. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

The compounds can be used as lotions, for example to prevent infectionof burns, by formulation with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant containing the antimicrobial polypeptides of theinvention is placed in proximity to the site of infection, so that thelocal concentration of active agent is increased relative to the rest ofthe body.

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with the compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 pg to 100milligrams per kg weight of subject per administration. A typical dosagemay be one tablet taken from two to six times daily, or one time-releasecapsule or tablet taken once a day and containing a proportionallyhigher content of active ingredient. The time-release effect may beobtained by capsule materials that dissolve at different pH values, bycapsules that release slowly by osmotic pressure, or by any other knownmeans of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

The use of liposomes as a delivery vehicle is one method of interest.The liposomes fuse with the cells of the target site and deliver thecontents of the lumen intracellularly. The liposomes are maintained incontact with the cells for sufficient time for fusion, using variousmeans to maintain contact, such as isolation, binding agents, and thelike. In one aspect of the invention, liposomes are designed to beaerosolized for pulmonary administration. Liposomes may be prepared withpurified proteins or peptides that mediate fusion of membranes, such asSendai virus or influenza virus, etc. The lipids may be any usefulcombination of known liposome forming lipids, including cationic orzwitterionic lipids, such as phosphatidylcholine. The remaining lipidwill be normally be neutral or acidic lipids, such as cholesterol,phosphatidyl serine, phosphatidyl glycerol, and the like.

For preparing the liposomes, the procedure described by Kato et al.,(1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids andlumen composition containing peptides are combined in an appropriateaqueous medium, conveniently a saline medium where the total solids willbe in the range of about 1-10 weight percent. After intense agitationfor short periods of time, from about 5-60 sec., the tube is placed in awarm water bath, from about 25-40° C. and this cycle repeated from about5-10 times. The composition is then sonicated for a convenient period oftime, generally from about 1-10 sec. and may be further agitated byvortexing. The volume is then expanded by adding aqueous medium,generally increasing the volume by about from 1-2 fold, followed byshaking and cooling. This method allows for the incorporation into thelumen of high molecular weight molecules.

Formulations with Other Active Agents

For use in the subject methods, the antimicrobial polypeptides of theinvention may be formulated with other pharmaceutically active agents,particularly other antimicrobial agents. Other agents of interestinclude a wide variety of antibiotics, as known in the art. Classes ofantibiotics include penicillins, e.g. penicillin G, penicillin V,methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.;penicillins in combination with beta-lactamase inhibitors,cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.;carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides;lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical;metronidazole; spectinomycin; trimethoprim; vancomycin; etc.

Anti-mycotic agents are also useful, including polyenes, e.g.amphotericin B, nystatin; 5-flucosyn; and azoles, e.g. miconazol,ketoconazol, itraconazol and fluconazol. Antituberculotic drugs includeisoniazid, ethambutol, streptomycin and rifampin. Cytokines may also beincluded in a formulation of the antimicrobial polypeptides of theinvention, e.g. interferon gamma, tumor necrosis factor alpha,interleukin 12, etc.

In Vitro Synthesis

The antimicrobial peptides of the invention may be prepared by in vitrosynthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example automatedsynthesizers by Applied Biosystems Inc., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids, particularly D-isomers (or D-forms) e.g.D-alanine and D-isoleucine, diastereoisomers, side chains havingdifferent lengths or functionalities, and the like. The particularsequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like.

Chemical linking may be provided to various peptides or proteinscomprising convenient functionalities for bonding, such as amino groupsfor amide or substituted amine formation, e.g. reductive amination,thiol groups for thioether or disulfide formation, carboxyl groups foramide formation, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein

Animal Feed

The present invention is also directed to methods for using thepolypeptides having antimicrobial activity in animal feed, as well as tofeed compositions and feed additives comprising the antimicrobialpolypeptides of the invention.

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants, such as cows, sheep andhorses. In a particular embodiment, the animal is a non-ruminant animal.Non-ruminant animals include mono-gastric animals, e.g. pigs or swine(including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys and chicken (including but not limited tobroiler chicks, layers); young calves; and fish (including but notlimited to salmon).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the antimicrobial polypeptide canbe fed to the animal before, after, or simultaneously with the diet. Thelatter is preferred.

In a particular embodiment, the antimicrobial polypeptide, in the formin which it is added to the feed, or when being included in a feedadditive, is well defined. Well-defined means that the antimicrobialpolypeptide preparation is at least 50% pure as determined bySize-exclusion chromatography (see Example 12 of WO 01/58275). In otherparticular embodiments the antimicrobial polypeptide preparation is atleast 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determinedby this method.

A well-defined antimicrobial polypeptide preparation is advantageous.For instance, it is much easier to dose correctly to the feed anantimicrobial polypeptide that is essentially free from interfering orcontaminating other antimicrobial polypeptides. The term dose correctlyrefers in particular to the objective of obtaining consistent andconstant results, and the capability of optimising dosage based upon thedesired effect.

For the use in animal feed, however, the antimicrobial polypeptide neednot be that pure; it may e.g. include other enzymes, in which case itcould be termed an antimicrobial polypeptide preparation.

The antimicrobial polypeptide preparation can be (a) added directly tothe feed (or used directly in a treatment process of vegetableproteins), or (b) it can be used in the production of one or moreintermediate compositions such as feed additives or premixes that issubsequently added to the feed (or used in a treatment process). Thedegree of purity described above refers to the purity of the originalantimicrobial polypeptide preparation, whether used according to (a) or(b) above.

Antimicrobial polypeptide preparations with purities of this order ofmagnitude are in particular obtainable using recombinant methods ofproduction, whereas they are not so easily obtained and also subject toa much higher batch-to-batch variation when the antimicrobialpolypeptide is produced by traditional fermentation methods.

Such antimicrobial polypeptide preparation may of course be mixed withother enzymes.

The term vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g. soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, and sorghum.

The antimicrobial polypeptide can be added to the feed in any form, beit as a relatively pure antimicrobial polypeptide, or in admixture withother components intended for addition to animal feed, i.e. in the formof animal feed additives, such as the so-called pre-mixes for animalfeed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives, e.g.premixes.

Apart from the antimicrobial polypeptide of the invention, the animalfeed additives of the invention contain at least one fat solublevitamin, and/or at least one water soluble vitamin, and/or at least onetrace mineral, and/or at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, aromacompounds, stabilisers, and/or at least one other enzyme selected fromamongst phytases EC 3.1.3.8 or 3.1.3.26; xylanases EC 3.2.1.8;galactanases EC 3_(—)2.1.89; and/or beta-glucanases EC 3.2.1.4.

In a particular embodiment these other enzymes are well defined (asdefined above for antimicrobial polypeptide preparations).

Examples of other antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Ovispirin such asNovispirin (Robert Lehrer, 2000), and variants, or fragments thereofwhich retain antimicrobial activity.

Examples of other antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Usually fat and water soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with an antimicrobial polypeptide ofthe invention, is an animal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g. vitamin K3.

Examples of water soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g. Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneantimicrobial polypeptide as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& Iooijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein or protein source as definedabove.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-10% fish meal; and/or 0-20% whey. Animal diets can e.g.be manufactured as mash feed (non pelleted) or pelleted feed. Typically,the milled feed-stuffs are mixed and sufficient amounts of essentialvitamins and minerals are added according to the specifications for thespecies in question. Enzymes can be added as solid or liquid enzymeformulations. For example, a solid enzyme formulation is typically addedbefore or during the mixing step; and a liquid enzyme preparation istypically added after the pelleting step. The enzyme may also beincorporated in a feed additive or premix.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, for example in the range of 5-30mg enzyme protein per kg animal diet.

The antimicrobial polypeptide may be administered in one or more of thefollowing amounts (dosage ranges): 0.01-200; or 0.01-100; or 0.05-100;or 0.05-50; or 0.10-10—all these ranges being in mg antimicrobialpolypeptide protein per kg feed (ppm).

For determining mg antimicrobial polypeptide protein per kg feed, theantimicrobial polypeptide is purified from the feed composition, and thespecific activity of the purified antimicrobial polypeptide isdetermined using a relevant assay (see under antimicrobial activity,substrates, and assays). The antimicrobial activity of the feedcomposition as such is also determined using the same assay, and on thebasis of these two determinations, the dosage in mg antimicrobialpolypeptide protein per kg feed is calculated.

The same principles apply for determining mg antimicrobial polypeptideprotein in feed additives. Of course, if a sample is available of theantimicrobial polypeptide used for preparing the feed additive or thefeed, the specific activity is determined from this sample (no need topurify the antimicrobial polypeptide from the feed composition or theadditive).

Signal Peptide and Propeptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to one or both of a firstnucleotide sequence consisting of nucleotides 1 to 60 of SEQ ID NO:1encoding a signal peptide consisting of amino acids −48 to −29 of SEQ IDNO:2 and a second nucleotide sequence consisting of nucleotides 61 to144 of SEQ ID NO:1 encoding a propeptide consisting of amino acids −28to −1 of SEQ ID NO:2, wherein the gene is foreign to the first andsecond nucleotide sequences.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The first and second nucleotide sequences may be operably linked toforeign genes individually with other control sequences or incombination with other control sequences. Such other control sequencesare described supra. As described earlier, where both signal peptide andpropeptide regions are present at the amino terminus of a protein, thepropeptide region is positioned next to the amino terminus of a proteinand the signal peptide region is positioned next to the amino terminusof the propeptide region.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade. In the following examples, the antimicrobialpolypeptide shown as amino acids 1 to 42 of SEQ ID NO:2 is referred toas “Eurocin”.

Example 1 cDNA library of Eurotium amstelodami

A cDNA library was prepared from E. amstelodami grown for 5 days onPotato dextrose agar (PDA). PolyA enriched RNA was purified, cDNA wassynthesized and the library made according to standard molecular biologyprocedures. A detailed protocol on the general process can be found inthe examples of international patent application WO 01/12794. The vectorused for cloning was pMhas5, which is shown in SEQ ID NO:8, and whichhas the following features:

TABLE 1 Features of vector pMhas5. Feature Location Description CDS 365-1156 Kanamycin resistance CDS 2232-2387 Beta galactosidase alphapeptide −10 signal 2189-2192 Shine Dalgarno promotor 2101-2189 Lacpromotor misc feature 626-650 KanP1 primer for BACE system

Example 2 Construction of an Aspergillus Expression Vector for Eurocin

The Eurocin encoding nucleotide sequence was amplified from the cDNAlibrary (see Example 1) in the following manner: 1 microliter of cDNA(approximately 10 nanogram of DNA) was used as template in a PCRreaction with Primer A and Primer B.

(SEQ ID NO: 4) Primer A: TCTTGGATCCACCATGCACTTCACCAAGGTCTCC(SEQ ID NO: 5) Primer B: TCTTCTCGAGTTAGAAAGAACAGGTGCAGGTAGG

10 pmole of each primer was used in a 50 microliter reaction volume.Annealing temperature was 55 degrees Celsius and extension at 72 degreesCelsius for 1 minute. A total of 35 cycles were run. The Expand HighFidelity PCR System (Roche) was used.

Aliquots of the PCR reaction were separated on a 4% agarose gel. Adistinct band was seen at a size of approximately 300 bp. The PCRfragment was digested with BamH1 and Xho1 which cut in the overhangsintroduced by the PCR primers. The digested fragments were isolated andcloned into BamH1-Xho1 digested pMT2786, an Aspergillus expressionplasmid based on the plasmid pMT2188, which is based on pCaHj527 (seExample 7 of international patent application WO 02/12472; and WO00/70064). The sequence of the insert was verified to encode the Eurocinsequence as identified above. The sequence of the PCR fragment is shownas SEQ ID NO:6.

The Aspergillus expression plasmid with the PCR product insert was namedpMT2935 and is shown as SEQ ID NO:7.

Example 3 Expression of Eurocin in Aspergillus

pMT2935 (see SEQ ID NO:7) was transformed into Aspergillus oryzae strainBECh2 (see international patent application WO 00/39322). 30transformants were re-isolated twice under selective and non-inducingconditions on Cove minimal plates with sucrose and acetamide. To testexpression of Eurocin, transformants were grown for 6 days at 30 degreescelcius in tubes with 10 ml YPM (2% peptone, 1% yeast extract, 2%maltose). Supernatants were run on NuPage 10% Bis-Tris SDS gels(Invitrogen) as recommended by the manufacturer with MES running bufferto allow separation in the low Mw range. The Aspergillus oryzaetransformants grew well even when induced for the expression of Eurocin.A distinct band of the size expected for Eurocin was seen in mosttransformants whereas this band was not seen in the untransformed hoststrains A. oryzae BECh2.

Example 4 Purification of Eurocin

The fermentation broth was filtered through a 0.22 μm Corning 431118filter and pH adjusted to 4.0 using formic acid. Filtering through a0.22 μm filter was repeated to remove additional precipitate afteradjusting pH. The conductivity was measured and adjusted toapproximately 10 mS cm⁻¹ by adding MQ-water (Millipore). The filtratewas then loaded onto a 12 ml Source 30S (Amersham Biosciences17-1273-01) column using an AKTA explorer HPLC instrument. Loadingbuffer was 50 mM formic acid, pH 4 (buffer A).

Eurocin was eluted using a gradient of 0-100% buffer B of 20 columnvolumes. Buffer B was 50 mM formic acid, 2 M sodium chloride, pH 4.0.The identity of the purified fractions was checked by MALDI-TOF MS(Voyager DE Pro instrument, Applied Biosystems) and the purity roughlychecked by SDS-PAGE (Invitrogen, NuPage 12% Bis-Tris/MES, stained withGel code Blue Stain Reagent, Pierce 24592)

Fractions containing the relevant compound (based on MALDI-TOF MS) werepooled and concentrated on an Amicon Ultra 5000 Da MWCO filter(Millipore UFC900524). Centrifugation was done at 4000 g forapproximately 30 minutes. The solution was then desalted by washingthree times with 0.01% acidic acid using the same filter andcentrifugation conditions.

The product was analyzed for correct sequence and purity by amino acidanalysis and MALDI-TOF MS. The measured mass of Eurocin was 4339 Da. Thetheoretical molecular weight is 4338.8 Da, average, based on amino acids1 to 42 of SEQ ID NO:2.

The stock solution was stored at −20° C.

Example 5 Evaluation of Antimicrobial Activity

Purified Eurocin was evaluated for antimicrobial activity in amicrodillution assay. The

Minimal Inhibitory Concentration (MIC) was determined following theNCCLS/CLSI guidelines from CLSI (Clinical and Laboratory StandardsInstitute).

Eurocin was tested at the following concentrations: 32; 16; 8; 4; 2; 1;0.50; 0.25; 0.13; and 0.06 μg/ml.

As shown in Table 2, Eurocin exhibits strong antimicrobial activityagainst a variety of ATCC strains.

TABLE 2 Antimicrobial activity of Eurocin MIC Strain (μg/ml)Staphylococcus aureus, ATCC 29213 16 Staphylococcus epidermidis, ATCC12228 16 Staphylococcus carnosus, ATCC 51365 8 Streptococcus pneumoniae,ATCC 49619 0.25 Streptococcus pyogenes, ATCC 12344 0.13 Enterococcusfaecium, ATCC 49624 16 Micrococcus luteus, ATCC 9341 0.5

Example 6 Using the HMM Files from the PFAM Database to Identify aDefensin

Sequence analysis using hidden markov model profiles (HMM profiles) maybe carried out either online on the Internet or locally on a computerusing the well-known HMMER freely available software package. Thecurrent version is HMMER 2.3.2 from October 2003.

The HMM profiles may be obtained from the well-known PFAM database. Thecurrent version is PFAM 16.0 from November 2004. Both HMMER and PFAM areavailable for all computer platforms from e.g. Washington University inSt. Louis (USA), School of Medicine (http://pfam.wustl.edu andhttp://hmmer.wustl.edu).

If a query amino acid sequence, or a fragment thereof, belongs to one ofthe following five PFAM families, the amino acid sequence is a defensinaccording to the present invention:

-   -   Defensin_beta or “Beta Defensin”, accession number: PF00711;    -   Defensin_propep or “Defensin propeptide”, accession number:        PF00879;    -   Defensin_(—)1 or “Mammalian defensin”, accession number:        PF00323;    -   Defensin_(—)2 or “Arthropod defensin”, accession number:        PF01097;    -   Gamma-thionin or “Gamma-thionins family”, accession number:        PF00304.

An amino acid sequence belongs to a PFAM family, according to thepresent invention, if it generates an E-value which is greater than 0.1,and a score which is larger or equal to zero, when the PFAM database isused online, or when the hmmpfam program (from the HMMER softwarepackage) is used locally.

When the sequence analysis is carried out locally using the hmmpfamprogram, it is necessary to obtain (download) the HMM profiles from thePFAM database. Two profiles exist for each family; xxx_ls.hmm for glocalsearches, and xxx_fs.hmm for local searches (“xxx” is the name of thefamily). That makes a total of ten profiles for the five familiesmentioned above.

These ten profiles may be used individually, or joined (appended) into asingle profile (using a text editor—the profiles are ASCII files) thatcould be named e.g. defensin.hmm. A query amino acid sequence can thenbe evaluated by using the following command line:

-   -   hmmpfam-E 0.1 defensin.hmm sequence_file    -   wherein “sequence_file” is a file with the query amino acid        sequence in any of the formats recognized by the HMMER software        package.

If the score is larger or equal to zero (0.0), and the E-value isgreater than 0.1, the query amino acid sequence is a defensin accordingto the present invention.

The PFAM database is further described in Bateman et al. (2004) “ThePfam Protein Families Database”, Nucleic Acids Research, Vol. 32(Database Issue) pp. D138-D141.

1. A method for killing or inhibiting growth of microbial cellscomprising contacting the microbial cells with an antimicrobial peptide,wherein the antimicrobial peptide is encoded by a polynucleotide whichhybridizes under high stringency conditions with (i) nucleotides 145 to270 of SEQ ID NO:1, (ii) the cDNA sequence contained in nucleotides 1 to270 of SEQ ID NO:1, or (iii) the complementary strand of (i) or (ii),wherein the high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 mg/ml sheared anddenatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours, followed by washingthree times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.
 2. Themethod of claim 1, wherein the antimicrobial peptide is encoded by apolynucleotide which hybridizes under very high stringency conditionswith (i) nucleotides 145 to 270 of SEQ ID NO:1, (ii) the cDNA sequencecontained in nucleotides 1 to 270 of SEQ ID NO:1, or (iii) thecomplementary strand of (i) or (ii), wherein the very high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA,and 50% formamide, following standard Southern blotting procedures for12 to 24 hours, followed by washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 70° C.
 3. A method for killing or inhibitinggrowth of microbial cells comprising contacting the microbial cells withan antimicrobial peptide, wherein the antimicrobial peptide has at least90% identity with amino acids 1 to 42 of SEQ ID NO:2.
 4. The method ofclaim 3, wherein the antimicrobial peptide has at least 95% identitywith amino acids 1 to 42 of SEQ ID NO:2.
 5. The method of claim 3,wherein the antimicrobial peptide has at least 96% identity with aminoacids 1 to 42 of SEQ ID NO:2.
 6. The method of claim 3, wherein theantimicrobial peptide has at least 97% identity with amino acids 1 to 42of SEQ ID NO:2.
 7. The method of claim 3, wherein the antimicrobialpeptide has at least 98% identity with amino acids 1 to 42 of SEQ IDNO:2.
 8. The method of claim 3, wherein the antimicrobial peptide has atleast 99% identity with amino acids 1 to 42 of SEQ ID NO:2.
 9. Themethod of claim 3, wherein the antimicrobial peptide comprises aminoacids 1 to 42 of SEQ ID NO:2.
 10. The method of claim 3, wherein theantimicrobial peptide consists of amino acids 1 to 42 of SEQ ID NO:2.